Epithelial antimicrobial proteins are evolutionarily ancient innate immune effectors. As key elements of intestinal mucosal defense, they likely play an important role in maintaining mutually-beneficial host-microbial relationships by restricting contact between resident microbes and mucosal surfaces. This idea is underscored by the fact that deficiencies in antimicrobial peptide expression are associated with inflammatory bowel disease (Wehkamp et al., 2004; Wehkamp et al., 2005), a chronic inflammatory disorder thought to be triggered by resident gut microbes. However, although cationic antimicrobial peptides such as defensins are well-characterized, the full repertoire of gut antimicrobial mechanisms remains undefined.
C-type lectins are proteins that contain carbohydrate recognition domains (CRDs) and bind selectively to specific carbohydrate structures, often in a Ca2+-dependent manner. They mediate a variety of functions including cellular adhesion, clearance of circulating proteins, and recognition of microbe-associated molecular patterns (reviewed in Drickamer et al., 1993). The Reg gene family encodes an extensive group of secreted proteins that contain conserved sequence motifs found in all C-type lectin CRDs. The family is so named because the first member to be identified was cloned from a cDNA library derived from regenerating pancreatic islets (Terazono et al., 1988). Subsequently, several members of this multigene family have been identified in mice and humans, and are grouped according to homology into four subfamilies: RegI, RegII, RegIII, and RegIV. Despite their similarities to well-characterized C-type lectins, the members of the Reg family have poorly defined functions and their carbohydrate ligands have not been clearly identified.
Members of the RegIII family are constitutively expressed at high levels in mouse and human gastrointestinal tissues. RegIIIα, β, and γ are expressed in mouse small intestine (Narushima et al., 1997), while human hepatocarcinoma-intestine-pancreas/pancreatitis associated protein (HIP/PAP) is made in human small intestine. RegIIIβ and γ expression levels increase dramatically in response to bacterial colonization and other inflammatory stimuli in mice (Ogawa et al., 2000; Ogawa et al., 2003; Keilbaugh et al., 2005). In addition, HIP/PAP expression is upregulated in the mucosal tissues of patients with inflammatory bowel disease Ogawa et al., 2003; Dieckgraefe et al., 2002). Despite these insights into the forces regulating RegIII protein expression, almost nothing is known about the biological functions of RegIII proteins or their role in disease.
An abundant source of purified recombinant mouse and human RegIII proteins is needed to delineate the role of RegIII proteins in intestinal biology and human disease. Human HIP/PAP has been purified previously from the milk of transgenic mice engineered to express the protein in mammary gland (Christa et al., 2000), and as a glutathione S-transferase (GST) fusion protein in an E. coli expression system (Christa et al., 1994). Although the transgenic approach yielded quantities of protein sufficient for crystallographic analysis (Abergel et al., 1999), this method is technically challenging, time-consuming, and expensive. The recombinant fusion protein procedure produced only microgram quantities of the GST-tagged protein (Christa et al., 1994). Thus, a simple expression and purification system is needed to generate large quantities of Reg proteins to further characterize their roles as antimicrobial proteins.